Inhibition by 2-Deoxy-D-Glucose of Synthesis of Saccharomyces

Home , Phosphoglucomutase

JOURNAL OF BACTROLOGY, Aug. 1972, p. 419-429 Vol. 111, No. 2 Copyright 0 1972 American Society for Microbiology Printed in U.S.A. Inhibition by 2-Deoxy-D-Glucose of Synthesis of Glycoprotein Enzymes by Protoplasts of Saccharomyces: Relation to Inhibition of Sugar Uptake and Metabolism'

S.-C. KUO AND J. 0. LAMPEN Institute of Microbiology, Rutgers University, 7he State University of New Jersey, New Brunswick, New Jersey 08903 Received for publication 17 April 1972

The synthesis of the glycoprotein enzymes, invertase and acid phosphatase, by protoplasts of Saccharomyces mutant 1016, is inhibited by 2-deoxy-D-glucose (2-dG) after a 20- to 30-min lag period under conditions (external sugar to 2-dG ratio of 40:1) which cause only a slight decrease in total protein synthesis. Formation of one intracellular enzyme, alpha-glucosidase, is also sensitive, but production of another, alkaline phosphatase, is unaffected. A nonme- tabolized glucose analogue, 6-deoxy-D-glucose, had no inhibitory effect. The total uptake of external fructose and maltose was decreased by 2-dG after a lag period of about the same duration as that before the inhibition of synthesis of enzymes or of mannan and glucan; during this time 2-dG was taken up by the protoplasts and accumulated primarily as 2-dG-6-phosphate (2-dG-6-P). Studies in vitro showed that 2-dG-6-P inhibits both yeast phosphoglucose isomerase and phosphomannose isomerase. The intracellular levels of the 6- phosphates of glucose, fructose, and mannose did not increase in the presence of 2-dG. We suggest that the high internal level of 2-dG-6-P blocks synthesis of the cell wall polysaccharides and glycoproteins in two ways. It directly inhibits the conversion of fructose-6-P to glucose-6-P and to mannose-6-P. At the same time, it restricts the transport of fructose and maltose into the cell; however, the continuing limited uptake of the sugars still provides sufficient energy for protein synthesis. The cessation of alpha-glucosidase synthesis is probably a result of depletion of the internal pool of maltose (the inducer). Our findings support the suggestion that restriction of synthesis of the carbohydrate moiety of glycoproteins reduces formation of the active enzyme.

2-Deoxy-D-glucose (2-dG), a potent inhibitor (15) and Liras and Gascon (27) reported that of the growth of yeast cells and of their fer- low levels of 2-dG selectively blocked the syn- mentation of sugars, has been the subject of thesis and secretion of mannan proteins (in- many studies. Its effects have usually been at- cluding invertase) by protoplasts under condi- tributed either to interference with the en- tions in which no significant inhibition of total trance of the fermentable sugars or to preven- protein synthesis was observed. tion of the synthesis of cell wall polysaccha- This communication describes the effect of rides by trapping uridine nucleotides as uri- 2-dG on the synthesis of two extemal enzymes, dine diphosphate 2-dG or by incorporating 2- the mannan proteins, acid phosphatase (EC dG into cell wall materials (5, 18, 21). As work 3.1.3.2) and invertase (EC 3.2.1.26) (6, 25, 28) with 2-dG progresses, it is becoming obvious and on two intracellular enzymes, alkaline that the glucose analogue has multiple effects phosphatase (EC 3.1.3.1) and alpha-glucosidase on cellular metabolism. Recently, Farkas et al. (EC 3.2.1.20) (17, 19, 28, 36), and the relation- ' A preliminary report of these findings was presented at ship between the intracellular level of 2-deoxy- the IV International Fermentation Symposium, Kyoto, Ja- glucose phosphates and the impairment of pan, March 1972. sugar uptake and metabolism. We also report 419 420 KUO AND) LAMPENI J. BACTERIOL. that 2-deoxyglucose-6-phosphate (2-dG-6-P) adding 1 ml of 1 M NaOH. Alkaline phosphatase was inhibits the conversion of fructose-6-P to glu- determined by incubating 1.6 ml of 1 M tris(hydroxy- cose-6-P and of mannose-6-P to fructose-6-P methyl)aminomethane (Tris)-hydrochloride (pH 8.9), by purified yeast phosphoglucose isomerase 0.1 ml of the test sample, and 0.3 ml of 0.04 M PNPP at 30 C; 1.0 ml of 1 M NaOH was added to (EC 5.3.1.9) and phosphomannose isomerase stop the reaction. The increase of absorbancy at 410 (EC 5.3.1.8), respectively. The implications of nm resulting from the liberation of p-nitrophenol these findings with respect to the mechanism was measured with a Beckman DB spectropho- of the inhibition by 2-dG of synthesis of cell tometer. One unit of phosphatase activity is the wall polysaccharides and glycoprotein enzymes amount of enzyme which liberates 1 Amole of nitro- by protoplasts are discussed. phenol (or Pi) per hr. Phosphoglucose isomerase was determined in a coupled enzyme assay by the conversion of fructose- MATERIALS AND METHODS 6-P to glucose-6-P, which was further converted to 6- Yeast strain, preparation of organisms, and phosphogluconic acid with glucose-6-phosphate de- protoplasts. The Saccharomyces mutant 1016 used hydrogenase and nicotinamide adenine dinucleotide in these experiments is maltose-positive. It was phosphate (NADP). The formation of NADPH was chosen because it produces a considerable amount of measured spectrometrically at 340 nm. The reaction invertase if the cells are grown on either maltose or a mixture (1.0 ml) consisted of 100,umoles of Tris-hy- low-hexose medium (24). For experiments involving drochloride buffer (pH 8.0), 1 umole of NADP, 5 ug acid and alkaline phosphatases, the cells from a 24- of glucose-6-phosphate dehydrogenase, 1 gg of yeast hr slant were transferred into 100 ml of Vogel's (40) phosphoglucose isomerase (Boehringer), and fruc- medium N with added vitamins (24) containing 0.05 tose-6-P as indicated. The experiments were con- M maltose, and were incubated for 14 to 15 hr at 28 ducted at room temperature. C on a reciprocating shaker. Protoplasts were pre- Phosphomannose isomerase was measured by the pared from these exponential-phase cells by the pro- formation of fructose-6-P from mannose-6-P. The cedure of Kuo and Lampen (24) and washed with 0.8 reaction mixture (1.0 ml) contained 100 gmoles of M sorbitol in the modified Vogel's medium N with Tris-hydrochloride buffer (pH 7.6), 0.035 Mig of yeast the phosphate omitted (0.8 M sorbitol medium) as phosphomannose isomerase (Sigma), and mannose- used by McLellan and Lampen (28). 6-P in the absence or presence of 2-dG-6-P as indi- For induction of alpha-glucosidase in the proto- cated. After incubation for 2 min at 28 C, the reac- plasts, the cells were grown in 0.2 M glucose. Proto- tion was terminated by removing 0.5 ml of sample plasts were prepared and washed with phosphate into 1.0 ml of Tris-hydrochloride buffer held at 95 C. buffer containing 0.8 M sorbitol as previously de- The amount of fructose-6-P formed was then meas- scribed (24). Alpha-glucosidase synthesis was then ured by the increase of absorbancy at 340 nm due to measured in a medium containing maltose as in- the reduction of NADP to NADPH after additions of ducer. phosphoglucose isomerase and glucose-6-phosphate Measurement of enzyme formation and secre- dehydrogenase. The results were quantitated by the tion by protoplasts. Washed protoplasts were sus- use of an intemal standard of fructose-6-P, with or pended at 5 x 101 per ml in 0.8 M sorbitol medium without 2-dG-6-P in order to correct for the slight with the indicated concentration of sugar as energy interference by 2-dG-6-P in the determination (see source and incubated at 30 C in a reciprocating below). shaker. Exoenzyme production usually began after a Determination of intracellular hexose-6-phos- delay of 30 to 60 min. The basis of this lag is consid- phate. Washed protoplasts were suspended at 5 x ered below in relation to Fig. 5, which shows a sim- 107 per ml in 0.8 M sorbitol medium with the indi- ilar delay in uptake of 2-dG. To measure enzyme cated concentration of fructose or maltose and incu- released into the medium, 1.0 ml of the incubation bated at 30 C for 40 min. The suspensions were then mixture was withdrawn at intervals and centrifuged centrifuged at 1,000 x g for 3 min. To the pellets at 1,000 x g for 3 min; 0.5 ml of the supernatant was added 2 ml of 7% perchloric acid to stop the fluid was pipetted into 0.5 ml of ice-cold water and reaction and to extract sugar phosphates. Extraction assayed for the enzyme activity. For determination proceeded for 30 min at room temperature, followed of total enzyme synthesis, samples were periodically by neutralization of the samples to pH 7.0 with 0.5 N removed from the mixture and transferred to chilled KOH. After rapid freezing and thawing, protoplast tubes containing ice-cold water to lyse the proto- debris and precipitated KClO, were removed by cen- plasts. trifugation at 3,000 x g at 4 C. The supernatant Enzyme assays. The assays for invertase and fluids were lyophilized and finally suspended in 2 ml alpha-glucosidase were described previously (24). of 0.05 M Tris-hydrochloride buffer (pH 7.6). The 6- Acid and alkaline phosphatases were assayed with p- phosphates of glucose, 2-dG, fructose, and mannose nitrophenyl phosphate (PNPP) as substrate as de- were assayed by NADPH formation in the usual sys- scribed by Torriani (37). For acid phosphatase, the tems containing NADP and glucose-6-phosphate assay mixture consisted of 1.5 ml of 0.2 M acetate dehydrogenase, phosphoglucose isomerase, and phos- buffer (pH 4.0), 0.3 ml of 0.04 M PNPP, and 0.2 ml of phomannose isomerase as appropriate. Measurement the samples to be assayed. After incubation at 30 C of the hexose phosphates in the presence of 2-dG-6-P for various times, the reaction was terminated by is complicated by the fact that this ester is also a VOL. 111, 1972 INHIBITION OF GLYCOPROTEIN SYNTHESIS 421 substrate of the glucose-6-phosphate dehydrogenase deoxy-D-glucose was generously provided by R. K. from yeast (11). However, the relative rates of oxida- Crane, Rutgers Medical School. tion of glucose-6-P and 2-dG-6-P are approximately 100:0.36 (11), and the affinity of the enzyme for 2- dG-6-P must be very low since this compound did RESULTS not detectably inhibit oxidation of glucose-6-P even Effect of at a ratio of 80:1. It was therefore possible to esti- orthophosphate on the concur- mate the levels of the hexose-6-phosphates by the rent formation of acid phosphatase, alka- initial rapid burst of NADPH formation, and the 2- line phosphatase, and invertase by proto- dG-6-P by the subsequent slow production of plasts. Numerous studies with yeast cells have NADPH (higher concentration of glucose-6-phos- shown that the formation of acid and alkaline phate dehydrogenase). Intracellular concentrations phosphatase is repressed by the presence of were calculated according to the method described inorganic phosphate (19, 34, 41). Yeast proto- by Cirillo (8). plasts can synthesize these enzymes when in- Amino acid incorporation into the trichloroa- cubated in phosphate-free medium with appro- cetic acid-precipitable protein fraction; uptake of priate concentrations of sugar as energy source fructose, 2-dG, and maltose. The procedures were those of Kuo and Lampen (24) except that Whatman (28). We found, however, that the rate of acid glass-fiber paper (2.4 cm; GF/A) was used instead of phosphatase synthesis by yeast protoplasts was Millipore membrane filters. Trichloroacetic acid- markedly stimulated by addition of 1 to 10 mM precipitable protein probably does not include much inorganic phosphate. The synthesis of the en- of the glycoprotein since external invertase is not zyme was eventually repressed by phosphate precipitated by 5% trichloroacetic acid (29). but only after 2 to 3 hr of incubation. The re- Chromatographic separation of metabolites of sults of an experiment on the concurrent syn- 2-dG. To characterize the metabolites of 3H-2-dG thesis of acid and alkaline phosphatases and of accumulated by protoplasts, 2 ml of an incubation invertase are shown in Fig. 1. Formation of mixture was filtered through a Whatman glass-fiber paper (GF/A). The protoplasts were washed twice both acid phosphatase and invertase was with 5 ml of ice-cold 0.8 M KCI and then extracted greatly enhanced by the presence of inorganic for 10 min in 4 ml of 70% ethanol at 95 C. Extracts phosphate, and the newly synthesized enzymes were allowed to cool for 30 min at room temperature, were secreted (Fig. 1A, B). Alkaline phospha- filtered through membrane filters (0.45-Mm pore size, tase synthesis was not stimulated by phos- Millipore Corp.), and concentrated in a stream of phate, and the enzyme was retained by the nitrogen at 35 C. The concentrated extracts were protoplasts (Fig. 1C). After 2 hr, alkaline phos- applied to Whatman 3 MM filter paper and devel- phatase formation was repressed and total ac- oped by descending chromatography in one of the tivity gradually declined. Thus, in the pres- following solvents: (i) ethanol-methyl ethyl ketone- ence of 5 mm phosphate, both acid phospha- 0.5 M morpholinium tetraborate, pH 8.6, in 0.01 M tase and invertase could be the ethylenediaminetetraacetate (70:20:30) (7); (ii) iso- synthesized by butyric acid-1 M NH4OH (5:3) (1); (iii) ethanol-1 M protoplasts after a lag of only about 1 hr. Alka- ammonium acetate, pH 3.8 (7.5:3.0) (31). The chro- line phosphatase formation could be studied matograms were cut into 1-cm strips, and the ra- during the first 2 hr. dioactivity was counted in a Packard Tri-Carb liquid Effect of 2-dG on the formation of acid scintillation spectrometer. and alkaline phosphatases, invertase, and Synthesis of mannan and glucan. Protoplasts (5 alpha-glucosidase. When protoplasts were x 107/ml) were incubated at 30 C in 0.8 M sorbitol incubated with 100 mm fructose in the pres- medium with 100 mm "C-fructose (0.2 ACi/ml of ence of 2.5 mm 2-dG (fructose to 2-dG ratio of suspension). At intervals, 1-ml samples were trans- 40:1), formation of the extemal ferred to ampoules containing 4 ml of 2.5 N NaOH. enzymes acid The extraction of mannan and glucan and the deter- phosphatase and invertase was severely inhib- mination of incorporated radioactivity followed the ited (Fig. 2A, B); but alkaline phosphatase procedure described by Northcote and Home (30) synthesis was not affected even when the 2-dG and Elorza and Sentandreu (12). was added at zero time (Fig. 2C). At the 40:1 Chemicals. All chemicals were reagent quality. L- ratio used, 2-dG did not significantly inhibit Threonine-U- 14C, fructose-U- I4C, and 2-dG-G-3H total protein synthesis as measured by the in- were nurchased from New England Nuclear Corp., corporation of "4C-threonine into the trichloro- Boston Mass; maltose-U- '4C, from Amersham/Searle acetic acid-insoluble protein fraction (Fig. 3). Co., Arlington Heights, Ill. NADP, phosphoman- To determine whether 2-dG inhibits pri- nose isomerase (yeast), alkaline phosphatase (calf intestinal mucosa), 2-dG, and 2-dG-6-P were ob- marily the formation of enzymes that are gly- tained from Sigma Chemical Co., St. Louis, Mo. coproteins, alpha-glucosidase was also investi- Glucose-6-phosphate-dehydrogenase (yeast) and gated since it is probably not a glycoprotein phosphoglucose isomerase (yeast) were products of (17). Alpha-glucosidase synthesis by proto- Boehringer Mannheim Co., Germany. The 3H-6- plasts is readily induced by 20 mm maltose in 422 KUO AND LAMPEN J. BACTERIOL.

4 w

N wIz

TIME (hr) FIG. 1. Effect of phosphate on the concurrent formation and secretion of acid phosphatase, invertase, and alkaline phosphatase by protoplasts. Protoplasts from cells grown in 0.05 M maltose were suspended at 5 x 107 per ml in 0.8 M sorbitol medium containing 100 mm fructose and were incubated at 30 C. At various time intervals, 0.5-ml samples of the incubation mixtures or of the supernatant fluids (1,000 x g, 3 min) were transferred into 0.5 ml of ice-cold water and assayed as described in Materials and Methods. Enzyme activity is expressed as units per milliliter of the incubation mixture. Symbols: (A) supernatant fluid, no added phosphate; (0) supernatant fluid, 5 mM phosphate added; (A) protoplast pellet, no added phosphate; (0) protoplast pellet, 5 mm phosphate added.

(A) ACID PHOSPHATASE (B) INVERTASE (C) ALKALINE PHOSPHATASE 05 650 67.5 -7

E_4 40 (6

5 I.- ~~~~~~~30 -4

FIG. 2. Effect of 2-dG on the concurrent synthesis of acid phosphatase, invertase, and alkaline phosphatase by protoplasts. Protoplasts were suspended at 5 x 107 per ml in 0.8 M sorbitol medium containing 100 mm fructose and were incubated at 30 C. Only total enzyme activity in incubation mixture was measured. Symbols: (O) no 2-dG added; (a) 2.5 mm 2-dG added to incubation mixture at zero time; (A) 2.5 mm 2-dG added at 90 min (1). the presence of an energy source (10 mm fruc- longer required (B, Fig. 4). If the maltose was tose) and an amino acid supplement (Fig. 4). also omitted, synthesis of alpha-glucosidase After 120 min of incubation, synthesis of and invertase ceased after a lag of about 15 alpha-glucosidase and maltose utilization had min. The addition of 0.5 or 1 mM 2-dG (mal- begun, and the additional fructose was no tose to 2-dG ratio of 40:1 or 20:1) to proto- VOL. 111, 1972 INHIBITION OF GLYCOPROTEIN SYNTHESIS 423 plasts actively synthesizing alpha-glucosidase and invertase inhibited further synthesis within 20 to 30 min. It has been shown that E alpha-glucosidase formation is sensitive to ca- IL tabolite repression by glucose (38, 39) and that o 40 inactivation of the maltose transport system and of alpha-glucosidase occurs when maltose- z grown cells are suspended in a medium con- 0 taining glucose (16, 32). However, under our 30 0 conditions (B, Fig. 4; 20 mM maltose) alpha- a. glucosidase synthesis by protoplasts of mutant 0 1016 was insensitive to glucose at concentra- 0 tions (0.5-1 mM) at which 2-dG completely 20 inhibited formation of the enzyme; in fact, approximately 50 mM glucose was required for z rapid repression. In addition, we have con- firmed the report by Liras and Gascon (27) 0 that synthesis of invertase by mutant FH4C, cr 10 which is highly resistant to hexose repression (26), is as sensitive to 2-dG as in a hexose-repressible strain (mutant 1016). Thus, catabolite repression does not appear to be a significant factor in the effects of 2-dG reported 0 60 120 ISO here. TIME (min) It was of interest to determine whether 6- FIG. 3. Threonine incorporation into the protein deoxy-D-glucose (6-dG) would inhibit the for- fraction in the presence of 2-dG. Experimental conditions as for Fig. 2; 0.1 umole (0.04 gCi) of 14C- mation of invertase or alpha-glucosidase. This threonine per ml was included in the suspension at analogue is taken up by the protoplasts but is zero time. Samples were removed at the times indi- not metabolized, as only free 6-dG can be re- cated and analyzed for the amount of radioactivity covered from protoplast extracts. 6-dG concen- incorporated (for details see Materials and Methods). trations as high as 20 mM (intracellular level of Symbols: (0) no 2-dG added; (0) 2.5 mM 2-dG the analogue was approximately 5 mM) showed added to incubation mixture at zero time; (A) 2.5 no impairment of the synthesis of invertase or mM 2-dG added at 90 min (1). alpha-glucosidase (data not presented in detail). at which point the rate of uptake decreased Metabolism of 2-dG by protoplasts. From sharply. The slow uptake of 2-dG is probably studies with intact yeast, Bauer and his asso- related to the 30- to 60-min lag usually seen ciates (4, 5, 13, 14) reported that 2-dG trans- before such protoplasts form substantial ported into the cell is transformed into various amounts of exoenzymes (Fig. 1, 2, and 4 [24]). metabolites including sugar phosphates, nu- It can be explained by assuming that freshly cleotide-2-dG, dideoxy-trehalose, and cell wall prepared protoplasts have suffered some materials. Since the conditions employed for membrane damage and are also in a relatively intact cells and for protoplasts differed consid- "energy-poor" state. Incubation with fructose erably, we examined the metabolism of this would permit membrane repair and the regen- glucose analogue by protoplasts in the pres- eration of metabolic energy stores to facilitate ence of a high level of fructose (100 mM). subsequent enzyme secretion and 2-dG uptake When 3H-2-dG and unlabeled fructose were and metabolism. It is noteworthy that the ini- added to freshly prepared protoplasts under tial uptake (first 3 min) of 2-dG (2.5 mM) in the conditions used to determine the sensi- the presence of fructose (100 mM) was much tivity of enzyme production (Fig. 2), the up- lower than when fructose was omitted from the take of 3H-2-dG (Fig. 5A) showed a biphasic suspension (Fig. SC). This is in agreement with time course. There was a slow uptake of 2-dG the previous reports that 2-dG and fructose during the first 30 min, followed by a faster share the same carrier for transport (9, 20, 23). linear phase. In contrast, 3H-2-dG added to To characterize the accumulated metabolites protoplasts which had been incubated with 100 of 2-dG, extracts of protoplasts incubated for mm fructose for 90 min (Fig. 5B) was promptly 120 min with fructose (100 mM) and 3H-2-dG taken up; within 10 min the intracellular con- were chromatographed in solvents i, ii, and iii centration of the labeled sugar reached 12 mM, (see Materials and Methods). A result typical 424 KUO AND LAMPEN J. BACTERIOL.

o Cl TS - %~zO.# - C,~~~~~~~~~~~~ 20 7E~~~~~~~~~~TM (min

0 w2A5B0200 10 5 9 3 0n 12 10 3 042050-1190 23

FIG. 4. Inhibition of aipha-glucosidase and invertase synthesis by 2-dG. Protoplasts from cells grown in 0.2 M glucose medium were suspended at 5 x 107 per ml in phosphate buffer containing 0.8 M sorbitol, 10 mM fructose, 0.5% Casamino Acids (Difco), and 20 mM maltose as inducer (arrow A). After 120 min of incubation (arrow B), when synthesis of alpha-glucosidase had begun, the protoplasts were centrifuged at 1,000 x g for 3 mmn,0washed~~with~0.8~ M~ sorbitol,~ ~~~~~~~2and suspended in medium containing Casamino Acids (Difco), but kicking fructose. Additions: (0), 20 mMw maltose; (0) 20 mM maltose, 0.5 mM-2dG; (M) 20 mM maltose, 1.0 mM 2-dG; (M) none. At various time intervals, 0.2-mI samples of the incubation mixtures were transferred into 0.8 ml of ice-cold water and assayed for alpha-glucosidase and invertase activity.

zo of both incubation procedures is shown in Fig. } 6. The extracts contained free 2-dG and two E B - 5.0 phosphorylated 0.8 C .O 15o and 2-exgucs-,-diphsphae(-dG- o ; tJ)~~~~~~~1, 6-P). This was concluded from the following FG6- o f a0i OX observations. (i) Treatment of the protoplast m glucos medium were suspended at x 107 per ml extracts with alkaline phosphatase (100rl,g per fructose, 7.5%Casamino Acids 2 ml of extract, 1 hr at 30 C) converted all radio- E mn (Difco)and fructose. Add : 'M~~~~~~IE2 active materials to free 3H-2-dG; (ii) radioac- ) none. At variostimenterval,02msample5.0 . tive spots A and B (Fig. 6) corresponded to ice-coldwateran10~~~~assayed fo alphaglucos e ad i tauhentiv2-dG arespectively; (iii) when the slowest running spot (C) was Co 30I 2I heated with 0.1 N HCl at 950C for 10 min and 0 30 6T6090 120 50 80 rechromatographed, all ofthe radioactivity was at the position of 2-dG-6-P. No 2-dG was detected in the glucan, mannan, or nucleotide FIG. 5. Uptake of 2-dG by protoplasts. Experi- sugar fractions. If incubation with 3H-2-dG mental conditions as forFig. 2, with 2.5 jimoles (2.0 continued for more than 2 hr, other minor gUCi) of 3H-2-dG per ml in the suspension. Samples soswr bando h hoaorm were removed at the times indicated, and uptake of otsere oined o t ch protograst the sugar was measured as described in Materials ncluding oneildentlfied as 2-dG-laPa Under and Methods. Symbols: (0) 2-dG added to incuba- these circumstances, only 1 to 2% of the total tion mixture at zero time (A); (0) 2-dG added at 90 radioactivity taken up could be detected in the mmn (B); (A) 2-dG added at 90 min to protoplasts glucan and mannan fractions. centrifuged and resuspended in fresh incubation Uptake of fructose and maltose and syn- mixture with fructoseF~~~~~~~~~omitted (C). thesis of glucancand mannan in the pres- VOL. 111, 1972 INHIBITION OF GLYCOPROTEIN SYNTHESIS 425 ence of 2-dG. It was important to determine C B A whether or not 2-dG leads to a selective (2dG-1,6-P) (2dG-6-P) (2dG) blocking of the synthesis (and secretion) of 25 n mannan protein as suggested by Farkas et al. (15). To a suspension of protoplasts in 14C- 20 | fructose (100 mM), 2-dG (2.5 mM) was added at " zero time or after 90 min of incubation as in , s. the preceding experiments. The total uptake of l "C-fructose and its incorporation into glucan 0 i and mannan by the protoplasts in the presence and absence of 2-dG are presented in Fig. 7. The addition of 2-dG did not immediately af- fect "IC-fructose uptake; subsequently, the / rate was significantly decreased (Fig. 7A). In- corporation of "4C-fructose into both glucan 0 10 20 30 40 and mannan was inhibited after a similar lag CENTIMETERS FROM ORIGIN (Fig. 7B and C). The results clearly indicate that polysaccharide synthesis is sensitive, al- FIG. 6. Chromatographic analysis of products of though no selective inhibition of mannan was 2-dG accumulation by protoplasts. Experimental detected. conditions as for Fig. 2; 2.5 mM 3H-2-dG was added Since alpha-glucosidase is not a mannan at 90 min, and incubation was continued for 40 min. protoplasts extract was prepared as described in protein, it is unlikely that the inhibition of its MaterialsTe and Methods. Samples were transferred to synthesis by 2-dGMG (Fig.y(Fig.4tnat4) tnesunnltis a result ofinOIterof inter- Whatman no. 3 MM paper and developed by de- ference with polysaccharide synthesis. "'C- scending chromatography in solvent no. i. The dried maltose uptake was therefore examined in the chromatograms were cut into 1-cm strips, and the absence and presence of 2-dG under the condi- radioactivity was determined.

(A) FRUCTOSE-"C UPTAKE (8) GLUCAN - 4C (C) MANNAN-14C 25- 30

20 6

-~~~~~~~ ~ ~ ~20 CL 5 -0C

10 a 1

0 60 120 ISO 0 60 (20 ISO 60 (20 (80 TIME (min)

FIG. 7. Uptake of fructose and synthesis of glucan and mannan by protoplasts in the presence and absence of 2-dG. Experimental conditions as for Fig. 2, with 100 ,moles (0.2 AsCi) of "4C-fructose per ml in the suspending medium; 2.5 mM 2-dG was added either at zero time or after 90 min incubation (1). Samples were removed at times indicated and analyzed for the total uptake of "4C-fructose and for the radioactivity incorporated into glucan and mannan. Symbols: (0) no 2-dG added; (0) 2.5 mM 2-dG added to incubation mixture at zero time; (A) 2.5 mm 2-dG added at 90 min. 426 KUO AND LAMPEN J. BACTERIOL. tions for alpha-glucosidase synthesis. Net up- erase. Since 2-dG-6-P was the predominant take was markedly inhibited starting about 20 intracellular form (Fig. 6) and this ester has min after addition of 2-dG (Fig. 8). From a been implicated as a metabolic inhibitor of previous study on maltose transport and fer- phosphoglucose isomerase from rat kidney (42), mentation, De la Fuente and Sols (10) con- its action on yeast phosphoglucose isomerase cluded that maltose transport is followed by and phosphomannose isomerase was investi- intracellular hydrolysis and phosphorylation of gated. The data for phosphoglucose isomerase the liberated glucose, with transport probably are plotted in Fig. 9 by the method of Line- the rate-limiting step in maltose fermentation. weaver and Burk; the presence of 2-dG-6-P af- Since net "4C-maltose uptake was significantly fected the apparent Km for fructose-6-P but decreased when the protoplasts were incubated not the Vmax of the enzyme, indicating that 2- with even a low level of 2-dG (Fig. 8), we ex- dG-6-P acts as competitive inhibitor. Table 1 amined whether any free glucose was released shows that 2-dG-6-P also inhibited the conver- into the suspending medium after intracellular sion of mannose-6-P to fructose-6-P by yeast hydrolysis by alpha-glucosidase. Outflow of phosphomannose isomerase. At ratios of 2-dG- glucose would be expected if glucose phospho- 6-P to mannose-6-P of 4: 1 and 8:1, the extent rylation, but not maltose transport, was im- of inhibition was, respectively, 30 and 49%. paired oy 2-dG or its metabolites. Induced It was important to determine the level of protoplasts (as in Fig. 4) were incubated with the hexose-6-phosphates in protoplasts and to or without 2-dG, and the suspending medium see whether they accumulated in the presence was tested for free glucose by the sensitive glu- of 2-dG as a result of the inhibition of the cose oxidase assay. None could be detected phosphohexose isomerases. As shown in Table (data not presented in detail). Thus, even in 2, the intracellular level of glucose-6-P was the presence of 2-dG, the permeation is still approximately 0.25 mM, and both fructose-6-P the limiting step in maltose utilization by pro- and mannose-6-P were less than 0.1 mM. In the toplasts. presence of 2-dG, the levels of these hexose Effect of 2-dG-6-P on yeast phosphoglu- phosphates did not increase, whereas the level cose isomerase and phosphomannose isom- of 2-dG-6-P reached 16 to 20 mM. It should be noted that these estimations by an enzymatic procedure of the internal level of 2-dG-6-P are consistent with the results obtained by extraction and separation of the metabolites of 3H-2-

IN, dG (Fig. 5 and 6, and text). o0

0 a-w

0I- cn 0 -J 4

0 120 140 160 180 200 220 TIME (min) FIG. 8. Effect of 2-dG on the uptake of '4C-maltose by protoplasts preincubated in maltose medium. Experimental conditions as for Fig. 4. After -5 -2 2 5 10 15 20 120 min of incubation with maltose and fructose, I/ Fructose-6-P.mM protoplasts were centrifuged and suspended in 20 mM "4C-maltose (0.5 gCi/ml) medium without or FIG. 9. Activity of phosphoglucose isomerase on with 2-dG. Samples were removed at the times indi- fructose-6-P in the absence and presence of 2-dG-6- cated and analyzed for total uptake of "4C-maltose. P. Assay procedure described in Materials and Symbols: (0) no 2-dG added; (0) 1.0 mM 2-dG Methods. Symbols: (0) no 2-dG-6-P added; (0) 1 added at 120 min. mM 2-dG-6-P; (0) 2 mM 2-dG-6-P. VOL. 111, 1972 INHIBITION OF GLYCOPROTEIN SYNTHESIS 427 TABLE 1. Inhibition of purified yeast mannan or mannan proteins since the forma- phosphomannose isomerase by 2-dG-6-Pa tion of glucan was inhibited to approximately the same degree as mannan. Most impor- 2-dG-6-P Mannose-6-P Fructose-6-P Inhibition tantly, 2-dG inhibited the total uptake of ex- (Mmoles) (MAmoles) (fmoles) (%) ternal sugars after a lag period of about the same duration as the lag before inhibition of 0 0.5 0.091 enzyme or polysaccharide synthesis. 1.0 0.5 0.080 11.0 Metabolism of 3H-2-dG by protoplasts under 2.0 0.5 0.063 30.5 our conditions resulted in the accumulation of 0 0.25 0.063 one major intermediate identified as 2-dG-6-P; 1.0 0.25 0.044 30.0 2.0 0.25 0.033 49.0 two minor radioactive spots on chromatograms were identified as free 2-dG and 2-dG-1,6-P. a The reaction mixture (1.0 ml) contained 50 No detectable radioactivity was found in the ,gmoles of Tris-hydrochloride buffer (pH 7.6), man- nucleotide sugar fraction or in mannan and nose-6-P and 2-dG-6-P as indicated, and yeast phos- glucan at a time when synthesis of polysaccha- phomannose isomerase (0.035 jAg of protein). The rides and mannan protein had been severely mixture was incubated at 28 C for 2.5 min. Fructose- inhibited. Free 2-dG had been reported to in- 6-P formed was assayed as described in Materials hibit yeast uridine diphosphate glucose-4-epi- and Methods. merase (3); however, this is probably not significant in the present investigations in which TABLE 2. Intracellular hexose-6-phosphate levels in external fructose is the carbon and energy protoplasts incubated with or without 2-dGa source. In rat diaphragm, 2-dG-6-P acts as a Glu- Fruc- Man- 2-dG- competitive inhibitor of phosphoglucomutase (22), but the activity of this enzyme in crude Sugar 2-dG cose- tose- nose- 6-P added 6-P 6-P 6-P (m extracts of protoplasts was not affected at a 2- (mM) (mM) (mM) (mM) dG-6-P to glucose-6-P ratio of 4:1. In addition, in of Fructose None 0.24 <0.1 <0.1 protoplasts incubated the presence 2-dG (100 mM) 2.5 mM 0.21 <0.1 <0.1" 16 and then lysed did not exhibit reduced phos- Maltose None 0.24 <0.1 <0.1 phoglucomutase activity. Nevertheless, inhibi- (20 mM) 1.0 mM 0.22 <0.lb <0.1b 21 tion of phosphoglucomutase in the intact protoplast cannot be excluded, since the ratios of a Protoplasts were incubated with fructose (as in 2-dG-6-P to glucose-6-P attained were higher Fig. 2) or maltose (Fig. 4) for 40 min with 2-dG as than 50: 1 (Table 2). Glucose-6-phosphate de- indicated. Hexose-6-phosphates were extracted with hydrogenase was not significantly inhibited by 7% perchloric acid and determined in the neutral- ized supematant fluids as described in Materials 2-dG-6-P (see Materials and Methods). Hexo- and Methods. kinase must also be relatively insensitive since IAdded fructose-6-P or mannose-6-P at 0.1 mM 2-dG-6-P accumulated in large quantities as a could be detected readily. metabolite of 2-dG, and the studies on maltose uptake showed that transport remained the rate-limiting step even in the presence of 2- dG. In contrast, our experiments clearly show DISCUSSION that 2-dG-6-P inhibits conversion of fructose- Yeast protoplasts provide an excellent 6-P to glucose-6-P by phosphoglucose isom- system for the study of enzyme synthesis and erase from yeast and of mannose-6-P to fruc- secretion; several,enzy'mes are actively synthe- tose-6-P by phosphomannose isomerase. De- sized under appropriate conditions and, on the spite the probable blocking of these important basis of available data, only those enzymes routes of hexose phosphate metabolism, there that are glycoproteins are liberated into the was no accumulation of the 6-phosphate esters suspending medium (24, 25). The present within the protoplasts. study confirms the reports by Farkas et al. (15) On the basis of these observations we sug- and Liras and Gascon (27) that 2-dG inhibits gest that the intracellular 2-dG-6-P is the the synthesis and secretion of invertase, a effective form of 2-dG and that it blocks poly- mannan protein, under conditions in which saccharide and glycoprotein synthesis in two total protein synthesis is relatively unaffected ways. It directly inhibits the conversion of (hexose to 2-dG ratio of 20: 1 or 40: 1). Forma- fructose-6-P to glucose-6-P and to mannose-6- tion of another mannan protein, acid phospha- P by the phosphohexose isomerases; concomi- tase, was also prevented. Nevertheless, the ac- tantly, it decreases the transport of fructose or tion of 2-deoxyglucose is not restricted to maltose into the cells. This proposed restric- 428 KUO AND LAMPEN J. BACTERIOL. tion of sugar transport by 2-dG-6-P is analo- wall materials. This probably accounts for gous to the earlier suggestions by Sols (33) and some of the other phenomena that have been Azam and Kotyk (2) that the intracellular reported (5, 18, 21, 35). level of glucose-6-P exerts a feedback control of hexose transport in yeast. The existence of a ACKNOWLEDGMENTS relatively direct effect of 2-dG-6-P on sugar We wish to thank F. R. Cano and V. P. Cirillo for stimu- lating discussions during the course of this investigation, transport is supported by the lack of any sig- and J. Vensel for skilled technical assistance. We also thank nificant accumulation of the 6-phosphates of R. Sentandreu for collaboration in the early experiments. fructose, glucose, or mannose in the treated This research was supported by Public Health Service protoplasts. grant AI-04572 from the National Institute of Allergy and Another indication of a critical role for re- Infectious Diseases. striction of transport is the observation that the sensitivity of glycoprotein synthesis to 2- LITERATURE CITED dG is essentially the same when mannose or 1. Anderson, J. S., P. M. Meadow, M. A. Haskin, and J. L. Strominger. 1966. Biosynthesis of peptidoglycan of glucose is used as the energy source in place of bacterial cell walls. 1. Utilization of uridine diphos- fructose (unpublished data). If the phospho- phate acetylmuramyl pentapeptide and uridine di- hexose isomerases are the only pertinent en- phosphate acetylglucosamine for peptidoglycan syn- zymes inhibited by 2-dG-6-P, these sugars thesis by particulate enzymes from Staphylococcus au- reus and Micrococcus lysodeikticus. Arch. Biochem. could still be converted to their 6-phosphates, Biophys. 116:487-515. thus possibly enabling synthesis of mannan 2. Azam, F., and A. Kotyk. 1969. Glucose-6-phosphate as and mannan proteins (from mannose) or of regulator of monosaccharide transport in baker's glucan (from glucose). The uniform sensitivity yeast. Fed. Eur. Biochem. Soc. Lett. 2:333-335. 3. Azam, F., S.-C. Kuo, and V. P. Cirillo. 1971. Production on the various sugars supports the concept of a of phenotypically epimeraseless yeast by L-arabinose. generalized inhibition of hexose transport. J. Bacteriol. 106:915-919. Since there is a continuing limited uptake of 4. Biely, P., and S. Bauer. 1968. The formation of guano- the sugars in the presence of 2-dG, there would sine diphosphate-2-deoxy-D-glucose in yeast. Biochim. Biophys. Acta 156:432-434. still be sufficient energy available for protein 5. Biely, P., Z. Kratky, J. Kovahk, and S. Bauer. 1971. synthesis, as demonstrated by the fact that Effect of 2-deoxyglucose on cell wall formation in alkaline phosphatase and total protein syn- Saccharomyces cerevisiae and its relation to cell thesis were not inhibited, but the large-scale growth inhibition. J. Bacteriol. 107:121-129. 6. Boer, P., and E. P. Steyn-Parve. 1966. Isolation and pu- production of glycoproteins or of the usual wall rification of an acid phosphatase from baker's yeast polysaccharides mannan and glucan would be (Saccharomyces cerevisiae). Biochim. Biophys. Acta halted. The cessation of alpha-glucosidase syn- 128:400-402. thesis after 20 to 30 min is probably caused by 7. Carminatti, H., and S. Passeron. 1966. Chromatography of sugar nucleotides in morpholinium borate, p. 108- the reduced uptake of maltose (the inducer) 111. In E. F. Neufeld and V. Ginsburg (ed.), Methods and the resulting depletion of the internal in enzymology, vol. VIII, complex carbohydrates. pool. Academic Press Inc., New York. The high sensitivity of glycoprotein syn- 8. Cirillo, V. P. 1962. Mechanism of glucose transport across the yeast cell membrane. J. Bacteriol. 84:485- thesis to 2-dG is consistent with the suggestion 491. by Farkas et al. (15) that restriction of syn- 9. Cirillo, V. P. 1968. Relationship between sugar structure thesis of the carbohydrate moiety of glycopro- and competition for the sugar transport system in teins reduces the synthesis of the active en- bakers' yeast. J. Bacteriol. 95:603-611. 10. De la Fuente, G., and A. Sols. 1962. Transport of sugars zyme; however, the mechanism by which this in yeast. II. Mechanisms of utilization of disaccha- takes place is unknown. It should be empha- rides and related glycosides. Biochim. Biophys. Acta sized that our studies have focussed on the ef- 56:49-62. fects of 2-dG at high sugar to inhibitor ratios, 11. Egyud, L. G., and W. J. Whelan. 1963. Substrate specificity of glucose-6-phosphate dehydrogenase from dif- conditions under which its action appears to ferent sources. Biochem..J. 86:11 P. be the most specific. Under these circum- 12. Elorza, M. V., and R. Sentandreu. 1969. Effect of cyclo- stances the important effects of 2-dG appear heximide on yeast wall synthesis. Biochem. Biophys. to be the inhibition of phosphohexose isomer- Res. Commun. 36:741-747. 13. Farkas, V., S. Bauer, and J. Zemek. 1969. Metabolism of ases and of sugar uptake. This inhibition is 2-deoxy-D-glucose in baker's yeast. m. Formation of concurrent with the accumulation of 2-dG-6-P. 2,-2'-dideoxy-a'-trehalose. Biochim. Biophys. Acta but preceeds the formation of detectable levels 184:77-82. of nucleotide-2-dG. In experiments with high 14. Farkas, V., A. Svoboda, and S. Bauer. 1969. Inhibitory effect of 2-deoxy-D-glucose on the formation of the concentrations of 2-dG or with prolonged incu- cell wall in yeast protoplasts. J. Bacteriol. 98:744-748. bation, one would expect the analogue to be 15. Farkas, V., A. Svoboda, and S. Bauer. 1970. Secretion of incorporated into nucleotide sugars and cell cell-wall glycoproteins by yeast protoplasts: effect of VOL. 111, 1972 INHIBITION OF GLYCOPROTEIN SYNTHESIS 429

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